Ž . Journal of Immunological Methods 226 1999 43–48 Application of a fluorometric assay to detect caspase activity in thymus tissue undergoing apoptosis in vivo Adrienne M. Gorman 1 , Ulrich A. Hirt 2 , Boris Zhivotovsky, Sten Orrenius, Sandra Ceccatelli ) Institute of EnÕironmental Medicine, DiÕision of Toxicology, Karolinska Institutet, S-171 77 Stockholm, Sweden Received 10 December 1998; received in revised form 18 February 1999; accepted 26 February 1999 Abstract To date, in vivo apoptosis within the thymus has been assessed using morphological criteria andror detection of a DNA ladder indicative of oligonucleosomal fragmentation of the DNA. Here, we have used a fluorometric method to investigate activation of the caspase protease family in the thymus following in vivo induction of apoptosis by injection of the synthetic glucocorticoid hydrocortisone. Cleavage of DEVD-MCA by caspase-3 and other group II caspases releases free MCA which can be detected fluorimetrically. We demonstrate a time-dependent increase in DEVD-MCA cleavage activity within this tissue indicating the activation of caspase-3 like enzymes. This activity was inhibited by the specific group II caspase inhibitor DEVD-CHO. The interpretation of increased caspase activity was confirmed by immunoblot analysis to reveal cleavage of the caspase-3 substrate, fodrin. In addition, agarose gel electrophoresis of the DNA yielded a ladder pattern, confirming the occurrence of apoptosis. This study demonstrates that DEVD-MCA cleavage activity may be a useful quantitative method for the analysis of apoptosis in thymus tissue. It is a relatively rapid procedure not requiring thymocyte isolation or gel electrophoresis and detects fairly early biochemical changes occurring during apoptosis. In the present study we have used this method to demonstrate the involvement of caspases in thymocyte apoptotic death induced in vivo by glucocorticoids. Thus, measurement of caspase activity in thymus tissue may have applications for studying the in vivo effects of immunotoxicants. q 1999 Elsevier Science B.V. All rights reserved. Keywords: DEVD; Fodrin; Glucocorticoids; Rat 1. Introduction One of the earliest reported properties of adrenal Ž glucocorticoids is the destruction of thymocytes see . Cohen, 1992 , detected both in vivo and in vitro ) Corresponding author. Tel.: q46-8-728-7586; Fax: q46-8- 329-041; E-mail: sandra.ceccatelli@imm.ki.se 1 Contributed equally with Ulrich A. Hirt to this work. 2 Contributed equally with Adrienne M. Gorman to this work. Ž Wyllie, 1980; Wyllie and Morris, 1982; Cohen and . Duke, 1984 . The mode of thymocyte death is by Ž . apoptosis Cohen, 1991; Williams, 1994 which is a controlled form of death characterised by shrinkage of the cell, condensation of the nucleus and eventual cellular fragmentation into apoptotic bodies. To date, the most commonly used indicators of apoptosis in thymus tissue are based on morphological analysis to identify shrunken cells with pyknotic nuclei andror DNA extraction with subsequent detection of a ‘DNA ladder’. The latter method is based on the observa- 0022-1759r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. Ž . PII: S0022-1759 99 00054-X